Experimental evidence indicates that the skeletal component of human serum alkaline phosphatase (AP) may be clinically useful as a marker for bone formation. However, the existing methods for distinguishing the skeletal isoenzyme of AP in serum are inadequate for general clinical application. The proposed research will allow for the development of clinically useful methods for accurately measuring the skeletal isoenzyme of AP in human serum. Our primary goal, however, is to determine the biochemical function of skeletal AP in vivo. The research will consist of two interrelated projects: (a) kinetic studies of the skeletal isoenzyme of AP in vivo and in vitro for a systematic evaluation of its biochemical role in skeletal metabolism; and (b) the preparation of specific antibodies against the skeletal isoenzyme of AP for use in immuno-assays. The isoenzyme will be purified from human and from animal sources for antibody preparation and for biochemical studies. The kinetic studies will (1) evaluate the phosphatase and phospho-transferase potentials of the skeletal isoenzyme of AP, (2) determine the ionic nature of the phosphoryl substrates for AP, and (3) assess the effects of inhibitors and activators of the isoenzyme in vitro (e.g. histamine, Pi, PPi, PEA, lipids, etc.) Kinetic analyses will also allow for a comparison of the effects of these isoenzyme-specific inhibitors of AP activity with their effects on the growth and development of cultured embryonic chick tibiae, osteoblasts, and osteoclasts, as well as on intact chicks. 32P-distribution, 45Ca accretion and resorption, and OH-proline uptake and release will be used to correlate changes in growth and development with changes in AP activity. Quantitative morphological analyses using tetracycline labels will also be performed, in collaboration with Dr. Gruber, in this laboratory. Chemical effectors of skeletal AP activity, including inhibitors and activators (see above) and amino acid specific chemical reagents, will also be evaluated as potential bases for isoenzyme determinations in human serum.